Method of constructing vectors for homologous recombination directed mutagenesis

ABSTRACT

The present invention provides a novel vector system and thereby a novel method for the simplified construction of recombinant vectors for directed mutagenesis. Said vector system is used to modify the eukaryotic genome, particularly of embryonic stem cells, at precise and predefined loci by the means of homologous recombination. Further more, said system finds its usage in the generation of new strategies for gene therapy and in the generation of genetically modified higher eukaryotic organisms.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to methods and vectors for makingspecific mutations in genes. More specifically, the invention relates tothe use of a vector system useful in modifying the eukaryotic genome,particularly of embryonic stem cells, at precise and predefined loci bythe means of homologous recombination.

[0003] 2. Description of the Related Art

[0004] Many different technologies have been described that lead tochromosomal alterations and thereby to a modification of the structureand/or expression of genes. One technique for targeted mutagenesis isbased on homologous recombination. The general methodologies oftargeting mutations into the genome of cells, and the process ofgenerating mouse lines from genetically altered embryonic stem (ES)cells with specific genetic lesions are well known (Bradley, 1991, Cur.Opin. Biotech. 2: 823-829).

[0005] A synthetic recombination vector which contains the geneticinformation of the targeted chromosomal locus recombines with thegenomic DNA after introduction into a cell. The vector usually containsa positive selection cassette which is flanked by the geneticinformation of the target locus to enrich for cells where the vectorsuccessfully recombines with the chromosomal DNA against the pool ofnon-recombinant cells. Stable integration leads to a long termresistance against certain pharmacological toxins. Examples are theresistance against G418, i.e., Geniticin, or Hygromycin by the action ofthe neomycin or hygromycin resistance genes, respectively. The positionof the positive selection cassette in the chromosomal vector DNA canfurther lead to a mutation of the gene as in classical knockoutexperiments, i.e., inactivation of gene function. Furthermore,inactivation or modification of regulatory elements of the target geneas well as of domains of the transcribed/translated gene product couldhave positive, negative or modulatory effects on future target genefunction.

[0006] Homologous recombination, that is carried out by the target DNAflanking the positive selection cassette, has to be selected against thebackground of unwanted non-homologous recombination that is thought tooccur over the vector ends. A negative selection cassette positioned atthe terminus of the vector will frequently be integrated by thenon-homologous recombination events. Stable expression of the negativeselection marker leads to cytotoxicity of otherwise non-cytotoxicagents. An example is the activated cytotoxicity of Gancyclovir by theaction of the Herpes Simplex virus thymidine kinase gene product(HSV-TK). The likelihood of obtaining a homologous recombination eventincreases with the size of the chromosomal vector DNA and is furtherdependent on the isogenicity between the genomic DNA of the vector andthe target cell.

[0007] The cloning of large chromosomal fragments (5,000-15,000 basepairs) of the target gene, the subcloning of this DNA into a bacterialplasmid vector, the mapping of the gene structure, the integration ofthe positive selection cassette into the vector and finally, theflanking of one or both homologous vector arms by a negative selectionmarker is a technically demanding task and generally requires longconstruction times (3-6 months). The construction of the recombinationvector itself is, therefore, most often the time limiting step intargeted mutagenesis experiments.

[0008] The prior art is deficient in the lack of effective means ofconstructing vectors for homologous recombination directed mutagenesis.The present invention fulfills this longstanding need and desire in theart.

SUMMARY OF THE INVENTION

[0009] An object of the present invention is to simplify theconstruction of vectors for the targeted mutation of genes by homologousrecombination in mammalian cells. These vectors facilitate theconstruction of specifically mutated cells, cell lines derived from theindividually mutated cells, and cells for the use in the production oftransgenic non-human animals.

[0010] Another object of the present invention is to provide a vectorsystem that avoids the drawbacks of conventional vector construction.

[0011] The present invention provide a vector system and a new procedurethat simplifies the construction of positive/negative selectioncassettes. This new method reduces the time required for theconstruction of such vectors from 3-6 months to about 14 days.

[0012] A particularly useful vector class contemplated by the presentinvention includes a linear lambda vector (lambdaKOS, i.e., knockoutshuttle) for the construction of genomic DNA libraries that comprises: astuffer fragment; an E.coli origin of replication; an antibioticresistance gene; a yeast origin of replication; a selectable markersuitable for use in yeast; a negative selectable marker suitable for usein mammalian cells; LoxP sequences for Cre recombinase directedconversion of said linear lambda phage vector into an E.coli/yeastshuttle plasmid.

[0013] An additional vector contemplated by the present invention is avector designed to specifically insert a positive selection cassetteinto cloned genomic DNA. The vector comprises an E.coli origin ofreplication; an antibiotic resistance gene; a selectable marker suitablefor use in yeast; a positive selectable marker suitable for use inmammalian cells; unique restriction endonuclease sequences flanking thepositive selectable marker so that the marker can be exchanged foranother positive selection marker; unique restriction endonucleasesequences for the excision of the positive selection cassette of thevector and restriction endonuclease sequences flanking the bacterial andyeast sequences to facilitate the removal of these sequences from thevector after yeast-mediated recombination into the genomic target siteof the shuttle plasmid.

[0014] An additional embodiment of the invention provides a method ofgenerating mutations at specific sites in cloned genomic DNA, comprisingthe steps of: cloning genomic DNA into a linear lambda vector; isolatinga clone of interest; converting the linear lambda vector containing thegenomic DNA of interest into a circular E.coli/yeast shuttle vector;identifying genomic DNA sequences intended for targeting of the positiveselection cassette; synthesizing deoxyoligonucleotides complementary tosequences flanking the site intended for targeting of the positiveselection cassette to the circular genomic shuttle vector; attaching thedeoxyolignucleotides to the positive selection cassette by ligation orPCR; co-transforming the E.coli/yeast shuttle vector containing thegenomic DNA of interest and the modified positive selection cassetteinto a yeast host cell, wherein an intact recombinant plasmid isselected for by culturing the yeast on an appropriate media by means ofthe gene products provided for by two independent yeast selectablemarkers of the E. coli/yeast shuttle vector and the positive selectioncassette and is obtained by performing homologous DNA recombinationbetween homologous regions of the vector containing genomic DNA and thesynthetically derived sequences which had been ligated onto the ends ofthe positive selection cassette thereby generating a new recombinantvector with the positive selection cassette inserted into the genomicDNA sequences. After yeast plasmid isolation, E.coli transformation andE.coli plasmid isolation, the E.coli/yeast sequences of the positiveselection cassette vector are removed, comprising the steps of:digesting the new recombinant vector with restriction endonucleasespecific for the unique restriction endonuclease sequences incorporatedinto the positive selection cassette vector which flank the E.coli/yeastsequences in the vector; ligating said digested vector; and identifyingligation products lacking the E.coli/yeast sequences desired to beremoved.

[0015] Also provided is a method for the production of mutated animalcells consisting of: linearization of a new recombinant vector at aunique restriction endonuclease sequence outside the sequences of thecloned genomic DNA; introduction of linearized DNA into an animal cell;and selection of transduced cells that express a positive selectablemarker. Preferably, the animal cells are embryonic stem cells or anyother cell type with the potential to generate an animal.

[0016] In yet another embodiment of the present invention, there isprovided a method for the production of non-human transgenic animalconsisting of: introduction of mutated animal cells into animal embryos;placement of embryos containing mutated cells into the uterus of afemale animal; and replacement of the nucleus of a fertilized egg withthe nucleus containing the modified genomic DNA.

[0017] Further provided are genomic and sub-genomic librariesconstructed in the linear lambda vector wherein the libraries arederived from genomic DNA isolated from the group consisting of animalcells, mammalian cells, rodent cells, murine cells and other highereukaryotic cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] So that the matter in which the above-recited features,advantages and objects of the invention, as well as others which willbecome clear, are attained and can be understood in detail, moreparticular descriptions of the invention briefly summarized above may behad by reference to certain embodiments thereof which are illustrated inthe appended drawings. These drawings form a part of the specification.It is to be noted, however, that the appended drawings illustratepreferred embodiments of the invention and therefore are not to beconsidered limiting in their scope.

[0019]FIG. 1 shows a diagrammatic representation of a linear lambdavector (lambdaKOS) that is generally representative of the type ofvector that may be used in the present invention.

[0020]FIG. 2 shows a diagrammatic representation of a E.coli/yeastshuttle vector that is generally representative of the type of vectorthat may be used in the present invention, and shows a general strategyfor the method of insertion of the positive cassette into the genomicDNA sequence.

[0021]FIG. 3 shows an example of positive selection cassette. Thecomplete URA/CAT-selection cassette can be removed by a digest with theendonuclease SfiI and replaced by any other desired positive selectioncassette.

[0022]FIG. 4 shows pMCS-1 constructed as a new plasmid, comprising: aninverted repeat of three endonuclease restriction sites (SfiI, SwaI andAscI) flanking three unique endonuclease restriction sites (BamHI, EcoRIand XhoI) for the insertion of positive selection cassette. Any of thecentral three sites can be used to insert a selection cassette.

DETAILED DESCRIPTION OF THE INVENTION

[0023] As used herein, “insertional mutagenesis system” shall refer to agenetic system which allows one to mutate a genomic locus by inserting agenetic marker with or without replacing genomic sequences at the siteof insertion.

[0024] As used herein, “targeted mutagenesis” shall refer to mutation ofa genetic locus by inserting or replacing parts of the locus with aselection cassette. The site of mutation is generally selected byhomologous recombination.

[0025] As used herein, “linear lambda vector” shall refer to DNAprepared from lambda phages which is linear as compared to DNA preparedfrom Cre-expressing bacteria which are infected with the lambda phagewhere the linear phage DNA is connected to circular plasmid DNA.

[0026] As used herein, “E.coli/yeast shuttle vector” shall refer to anyplasmid which contains replication origins for plasmid survival, i.e.,multiplication and selection in E.coli and yeast cells.

[0027] As used herein, “Cre recombinase” shall refer to the activity ofthe Cre recombinase protein, which recombines between short stretches ofDNA comprising two LoxP elements.

[0028] As used herein, “lambdaKOS genomic library” shall refer to acollection of lambda phages which represent, by cumulative content ofgenomic DNA, either a total mammalian genome or a specified fractionthereof.

[0029] As used herein, “negative selection vector” shall refer to avector containing a gene active in mammalian cells which allows thekilling of such cells carrying the vector.

[0030] As used herein, “Cre/LoxP recombination system” shall refer to asystem where the expression and activity of Cre-recombinase proteinleads to a recombination effort between two cognate LoxP sequences.

[0031] The present invention describes a method of DNA vectorconstruction to substantially improve the engineering of targetedmutation of genes by homologous recombination in mammalian cells. In oneembodiment, the present invention provides an insertional mutagenesissystem useful in constructing vectors for the targeted replicationmutagenesis of mammalian cells by homologous recombination in which themutagenesis system comprises: (a) a linear lambda vector for the cloningof genomic DNA to be flanked by negative selection markers; and (b) avector for the insertion of a positive selection cassette into clonedgenomic DNA.

[0032] The particularly unique features of this methodology include theconstruction of genomic DNA libraries in vectors with negativelyselectable markers flanking the cloning sites and replication originsfor E.coli and yeast cells to insert a positive selection cassette intothe cloned genomic DNA sequences by homologous recombination in yeastdirected by synthetic DNA sequences ligated onto the ends of a yeastreplication deficient positive selection cassette and identical to thetargeted genomic DNA sequences. This methodology simplifies theconstruction of positive/negative selection cassettes and furthermorereduces the time required for the construction of such vectors from 3-6months to about 14 days.

[0033] A linear lambda vector (lambdaKOS) system was invented hereinbased on lambda phage cloning which allows the construction ofrepresentative genomic libraries of essentially every eukaryotic genome.This vector of the present invention comprises a stuffer fragment of DNAflanked by restriction endonuclease sequences to facilitate thereplacement with and thereby the cloning of genomic DNA; an E.coliorigin of replication; an antibiotic resistance gene; a yeast origin ofreplication; a selectable marker suitable for use in yeast; negativeselectable markers suitable for use in mammalian cells; a direct repeatof recombinase sequences for recombinase directed conversion of thelinear lambda phage vector into an E. coli/yeast shuttle plasmid.Preferably, this vector or any variants thereof are created by the useof different negative selectable markers. In this insertionalmutagenesis system, the recombinase sequence and correspondingrecombinase are selected from the group consisting of LoxP sequences-Crerecombinase and Frt sequences-Flp recombinase or any similar substituteas would be readily known to one having ordinary skill in this art.

[0034] An additional vector, i.e., the positive selection cassettevector, contemplated by the present invention is a vector designed tospecifically insert a positive selection cassette into cloned genomicDNA. This vector comprises a plasmid origin of replication; anantibiotic resistance gene; a selectable marker suitable for use inyeast; a positive selectable marker suitable for use in mammalian cells;unique restriction endonuclease sequences flanking the positiveselectable marker so that the marker can be exchanged for anotherpositive selection marker; a unique restriction endonuclease sequencefor the linearization of the vector and restriction endonucleasesequences flanking the bacterial and yeast sequences to facilitate theremoval of these sequences from the vector. Preferably, the positiveselection cassette vector or any variants thereof are created by the useof a different positive selection marker.

[0035] An additional embodiment of the invention provides a method ofgenerating mutations at specific sites in cloned genomic DNA, comprisingthe steps of: cloning genomic DNA into the linear lambda vector;isolating a clone of interest; converting the linear lambda vectorcontaining the genomic DNA of interest into a circular E.coli/yeastshuttle vector by infecting a Cre recombinase expressing bacterialstrain; identifying genomic DNA sequences intended for targeting of thepositive selection cassette; synthesizing deoxyoligonucleotidescomplementary to sequences flanking the site intended for targeting ofthe positive selection cassette to the circular E.coli/yeast shuttlevector; attaching the synthetic deoxyoligonucleotides to the positiveselection cassette by ligation or PCR (see Example 2); co-transformingsaid E. coli/yeast shuttle vector containing the genomic DNA of interestand the modified positive selection cassette into a yeast host cell,wherein an intact recombinant plasmid is selected for by culturing yeaston an appropriate media by means of the gene products provided for bythe yeast selectable markers of the E.coli/yeast shuttle vector and thepositive selection cassette and is obtained by performing homologous DNArecombination between homologous regions of the vector containinggenomic DNA and the synthetically derived sequences which had beenligated onto the ends of the positive selection cassette therebygenerating a new recombinant vector with the positive selection cassetteinserted into the genomic DNA sequences. Preferably, the E.coli/yeastsequences of the positive selection cassette vector are removed,comprising the steps of: digesting the new recombinant vector withrestriction endonuclease specific for the unique restrictionendonuclease sequences incorporated into the positive selection cassettevector which flank the E.coli/yeast sequences in the vector; ligatingthe digested vector; and identifying ligation products lacking theE.coli/yeast sequences desired to be removed.

[0036] The present invention is also directed to a method for theproduction of mutated animal cells consisting of: linearizing therecombinant vector at a unique restriction endonuclease sequence outsidethe sequences of the cloned genomic DNA; introduction of the linearizedDNA into an animal cell; and selection of transduced cells that expressthe positive selectable marker. Preferably, the animal cells areembryonic stem cells.

[0037] The present invention is also directed to a method for theproduction of non-human transgenic animal consisting of: introduction ofthe mutated animal cells into animal embryos; placement of the embryoscontaining the mutated cells into the uterus of a female animal; andreplacement of the nucleus of a fertilized egg with the nucleuscontaining the modified genomic DNA.

[0038] The present invention is further directed to genomic andsub-genomic libraries constructed in the linear lambda vector whereinsaid libraries are derived from genomic DNA isolated from the groupconsisting of animal cells, mammalian cells, rodent cells, murine cellsand other higher eukaryotic cells.

[0039] The invention when compared to other techniques of homologousrecombination vector construction offers the following advantages:First, the recombinant phage library has to be prepared only once forthe particular genome of interest and can afterwards be amplified as aphage library in bacteria a million fold. Secondly, every gene locus isautomatically ready for isolation with any gene probe of interest.Thirdly, the phage system allows the propagation of large chromosomalDNA which assists successful homologous recombination. Fourthly, asimple transfection of a Cre recombinase expressing bacterial straincreates a shuttle plasmid in which the genomic DNA is automaticallyflanked by negative selection cassettes. Fifthly, the shuttle plasmidnot only allows the propagation in E. coli but also in yeast for whichit can be selected for (TRP1) after successful transformation. Finally,the introduction of the positive selection cassette follows a publishedprocedure (Nucleic Acid Res. 24:4594-4596) but is further improved bythe herewith described and newly developed positive selection cassetteand pMCS-1 which simplify the construction of variants.

[0040] In accordance with the present invention there may be employedconventional molecular biology, microbiology, immunology and recombinantDNA techniques within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Maniatis, Fritsch &Sambrook, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning:A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcriptionand Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal CellCulture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

[0041] The following terms shall have the definitions set out below. A“DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in either single stranded form,or a double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

[0042] A DNA “coding sequence” is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

[0043] The term “oligonucleotide” or “deoxyoligonucleotide” as usedherein is defined as a molecule comprised of two or more ribonucleotidesor deoxyribonucleotides, preferably more than three. Its exact size willdepend upon many factors which, in turn, depend upon the ultimatefunction and use of the oligonucleotide.

[0044] The term “primer” as used herein refers to an oligonucleotide,whether occurring naturally as in a purified restriction digest orproduced synthetically, which is capable of acting as a point ofinitiation of synthesis when placed under conditions in which synthesisof a primer extension product, which is complementary to a nucleic acidstrand, is induced, i.e., in the presence of nucleotides and an inducingagent such as a DNA polymerase and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, the sourceof primer and the method used. For example, for diagnostic applications,depending on the complexity of the target sequence, the oligonucleotideprimer typically contains 15-25 or more nucleotides, although it maycontain fewer nucleotides.

[0045] As used herein, the term “PCR” refers to the polymerase chainreaction that is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202to Mullis, as well as other improvements known in the art.

[0046] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to bacterial enzymes, each of which cutdouble-stranded DNA at or near a specific nucleotide sequence.

[0047] The labels most commonly employed for these studies areradioactive elements, enzymes, chemicals which fluoresce when exposed toultraviolet light, and others. A number of fluorescent materials areknown and can be utilized as labels. These include, for example,fluorescein, rhodamine, auramine, Texas Red, AMCA blue and LuciferYellow. A particular detecting material is anti-rabbit antibody preparedin goats and conjugated with fluorescein through an isothiocyanate.

[0048] The following examples are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

EXAMPLE 1 Creation of Linear Lambda Vector (LambdaKOS)

[0049] A vector system was created based on lambda phage cloning whichallows the construction of representative genomic libraries ofessentially every eukaryotic genome. The linear lambda vector(lambdaKOS) has the following features from left to right (see FIG. 1):cos end and left arm of the phage, synthetic LoxP fragment, HSV-tk gene,endonuclease recognition sites BamHI and SalI, stuffer fragment,endonuclease recognition sites SalI and BamHI, HSV-tk gene, plasmidvector (bacterial origin of replication, β-lactamase gene for ampicillinresistance), endonuclease recognition sites NotI, yeast origin ofreplication 2 micron, yeast auxotrophic TRP1 gene, synthetic LoxPfragment (same relative orientation as the previous one) and the rightarm of the phage and cos end. DNA prepared from the lytic growing phagecan be digested by the endonucleases SalI and BamHI and the stufferfragment replaced by genomic DNA partially digested by the endonucleaseSau3AI. A several fold representative genomic library of a eukaryoticgenome can thereby be established. Every genomic fragment (10,000-15,000base pairs) is automatically flanked by a negative selection cassette oneither site. Since the genomic library can be several fold redundant,virtually every genomic locus is represented by a lambdaKOS phage.Genomic clones of interest can be screened by classical filterhybridization of the lambda phage library. Infection of aCre-recombinase expressing bacterial strain with a chosen phage cloneleads to an automatic Cre mediated recombination of the lambda phageinto a high copy E.coli/yeast shuttle plasmid (pKOS).

EXAMPLE 2 Insertion of Positive Selection Cassette (URA3/CAT-SelectionCassette)

[0050] The insertion of the positive selection cassette(URA3/CAT-selection cassette) into the genomic DNA of pKOS is carriedout by cotransfection of the cassette with the particular pKOS plasmidinto yeast and selection for complementation of both auxothrophicrequirements of the TRP/URA3-deficient yeast strain. The particularyeast strain used in this system requires the gene products of URA3 andTRP1 to survive and grow on selection plates. The TRP1 gene is deliveredby the pKOS plasmid. The complementary URA3 gene is cotransfected aspart of the replication deficient positive selection cassette.

[0051] The URA3/CAT-selection cassette for cloning purposes is part of ahigh copy bacterial plasmid and has the following features (see FIG. 3):endonuclease recognition sites SfiI, BamHI, XhoI, SwaI and AscI, a yeastactive URA3 gene, a bacterial active chloramphenicol acetyltransferasegene (CAT) gene and endonuclease recognition sites SwaI, AscI and SfiI.Any positive selection marker (e.g. neomycin or hygromycin expressioncassettes) can be cloned into the endonuclease recognition sites BamHIand XhoI of the URA3/CAT-selection cassette prior to the yeasttransfection.

[0052] Digestion of the URA3/CAT-selection cassette with theendonuclease SfiI will generate incompatible overhangs (see FIG. 2) towhich synthetic double stranded oligonucleotides can be ligated. Thesequences of the left and right oligonucleotides have to match theflanking sites of the desired integration site in the genomic DNA of theshuttle plasmid. Generally 40 base pairs of homology on both sites aresufficient for a successful integration of the positive selectioncassette into the desired integration site of the pKOS plasmid. A verysimple procedure to attach the 40 base pairs of homology is possible bythe polymerase chain reaction (PCR) using oligonucleotide primers withthe following features from 5′ to 3′: Primer A: 40 base pairs ofhomology with the 5′ flanking sequence of the target locus and 15-20base pairs of the 5′ end of the positive selection cassette. Primer B:40 base pairs of homology with the 3′ flanking sequence of the targetlocus (opposite strand) and 15-20 base pairs of the 3′ end of thepositive selection cassette.

[0053] The generated URA3/CAT-selection cassette amplicon can becontransfected into the yeast strain with the pKOS plasmid of interestusing standard procedures.

EXAMPLE 3 Further Selection for Homologous Recombinant Vectors

[0054] After successful cotransfection of the yeast replicationincompetent positive selection cassette with the replication competentE.coli/yeast shuttle plasmid (pKOS), only the yeast cells survive on-URA/-TRP selection plates where the positive selection cassetterecombines with the shuttle plasmid. The plasmids are recovered from theyeast cells and transferred into E.coli which allows a further selectionby plating on chloramphenicol and ampicillin containing plates usingstandard procedures. Digestion of the plasmids with the endonucleaseAscI or SwaI and re-ligation eliminates the URA3/CAT part of thecompleted pKOS recombination vector which can be selectively grown inbacteria in ampicillin containing media. Alternatively, the completeURA/CAT-selection cassette can be removed by a digest with theendonuclease SfiI and replaced by any other desired positive selectioncassette.

[0055] In order to improve this replacement procedure, a new plasmid wasconstructed, pMCS-1, which has the following features (see FIG. 4): aninverted repeat of three endonuclease restriction sites (SfiI, SwaI andAscI) flanking three unique endonuclease restriction sites (BamHI, EcoRIand XhoI) for the insertion of positive selection cassettes for laterreplacement of the URA3/CAT cassette from pKOS after yeast mediatedrecombination. Any of the central three sites can be used to insert aselection cassette, e.g., a neomycin, and any of the flanking sites canbe used to swap the selection cassette with the pKOS inserted URA3/CATcassette using the respective endonuclease restriction sites. Finally,for the gene targeting in mammalian cells via homologous recombination,the completed vector is linearized using the endonuclease NotI prior totransfection into the mammalian cells.

EXAMPLE 4 Generation of LambdaKOS Genomic Libraries

[0056] A lambdaKOS genomic library from the mouse inbred strain LEX-1has been successfully generated with a 10 fold representation of themouse genome and used successfully several times to recover pKOS clonesof the desired genomic loci (6 of 6 independent trials) for homologousvector construction. In all cases the planned vector construction wascarried out using the above discussed procedures and all insertions ofthe URA3/CAT-selection cassette were proceeded without any improperrecombination events in yeast which would generate useless pKOSconstructs. The proper recombination carried out by the yeast systemleading to the integration of the positive selection cassette into thepKOS plasmid is, in itself, a surprise since pKOS carries two identicalHSV-thymidine kinase (HSV-TK) cassettes which are a perfect substratefor a recombinant process. Since the two HSV-TK cassettes areconstructed in pKOS with an inverted orientation, any recombinationtaking place between these two cassettes only leads to a inversion ofintervening DNA but not to a deletion or other deleterious effects onthe pKOS plasmid.

[0057] LambdaKOS genomic libraries or sublibraries are generally, butnot exclusively, generated by cloning random fragments of genomic DNAbetween the phage arms. It is usually important to obtain sequenceinformation from the inserted fragment ends in order to generate outsideprobes to analyze the mutated locus and to analyze and compareindividual pKOS clones. Since the genomic insert is flanked on bothsites by the HSV-TK cassettes, any pKOS sequencing primer orientedtowards the insert would generate useless information. The constructionof lambdaKOS allows the generation of two oligonucleotide primers whichcontain and reflect a single base pair different between the 5′ end ofthe two HSV-TK cassettes:

KOS-1: 5′-accacactgctcgaggat

KOS-2: 5′-accacactgctcgacgga

[0058] Both primers allow a direct sequencing of both genomic insertends, respectively.

EXAMPLE 5

[0059] The invented vector system consists of a completely newconstructed lambda phage (lambdaKOS, i.e., knockout shuttle) whichallows one to clone the complete genome of a eukaryotic organism in anegative selection vector to build a representative genomic library.This means every target gene of that genome is accessible for anyplanned mutation after one single cloning step (i.e., the generation ofthe genomic library) which has to be carried out only once for anindividual eukaryotic cell. The phage, containing the desired genomiclocus, can be isolated from the library which can easily be amplifiedand used for future clone isolation from other genomic loci. After anautomatic subcloning step mediated by the Cre/LoxP recombination system,an E.coli/yeast shuttle plasmid (pKOS) can be obtained. The positiveselection cassette is inserted into the plasmid by site-specifichomologous recombination in yeast.

[0060] The invention as compared to other techniques of homologousrecombination vector construction has the following advantages: therecombinant phage library has to be prepared only once for theparticular genome of interest and can afterwards be amplified as a phagelibrary in bacteria a million fold. Automatically, every gene locus isready for isolation with any gene probe of interest. The phage systemallows the propagation of large chromosomal DNA which assists successfulhomologous recombination. A simple transfection of a Cre recombinaseexpressing bacterial strain creates a shuttle plasmid in which thegenomic DNA is automatically flanked by negative selection cassettes.The shuttle plasmid not only allows the propagation in E.coli but alsoin yeast for which it can be selected for (TRP1) after successfultransformation. The introduction of the positive selection cassettefollows a published procedure (Nucleic Acid Res. 24: 4594-4596) but isfurther improved by the herewith described newly developed positiveselection cassette and pMCS-1 plasmid which simplify the construction ofvariants.

[0061] Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents an publications are herein incorporateby reference to the same extent as if each individual publication wasspecifically an individually indicated to be incorporated by reference.

[0062] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, anspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1 2 1 18 DNA artificial sequence Oligonucleotide primer from lambdaKOSwhich allows the direct sequencing of one of the genomic insert ends. 1accacactgc tcgaggat 18 2 18 DNA artificial sequence Oligonucleotideprimer from lambdaKOS which allows the direct sequencing of the othergenomic insert end. 2 accacactgc tcgacgga 18

What is claimed is:
 1. An insertional mutagenesis system useful inconstructing vectors for targeted mutagenesis of mammalian cells byhomologous recombination, said insertional mutagenesis systemcomprising: (a) a linear lambda vector for cloning of genomic DNAflanked by negative selection markers; and (b) a vector for insertion ofa positive selection cassette into cloned genomic DNA.
 2. Theinsertional mutagenesis system of claim 1, said linear lambda vectorcomprising: (a) a stuffer fragment of DNA flanked by restrictionendonuclease sequences to facilitate the cloning of genomic DNA; (b) anE.coli origin of replication; (c) an antibiotic resistance gene; (d) ayeast origin of replication; (e) a selectable marker suitable for use inyeast; (f) a negative selectable marker(s) including a promoter elementoperatively positioned 5′ to said negative selectable marker and apolyadenylation site operatively positioned 3′ to said negativeselectable marker. (g) a direct repeat of recombinase sequences forrecombinase directed conversion of the linear lambda phage vector intoan E. coli/yeast shuttle plasmid.
 3. The insertional mutagenesis systemof claim 2, wherein said recombinase sequence and correspondingrecombinase are selected from the group consisting of LoxP sequences-Crerecombinase and Frt sequences-Flp recombinase.
 4. The insertionalmutagenesis system of claim 1, said vector for the insertion of apositive selection cassette comprises: (a) an E.coli origin ofreplication; (b) an antibiotic resistance gene; (c) a selectable markersuitable for use in yeast; (d) a positive selectable marker, wherein apromoter element is operatively positioned 5′ to said positiveselectable marker and a polyadenylation site operatively positioned 3′to said positive selectable marker; (e) unique restriction endonucleasesequences flanking the positive selectable marker so that said markercan be exchanged for another positive selection marker; (f) uniquerestriction endonuclease sequences for the excision of the positiveselective cassette prior to ligation of synthetic deoxynucleotides tothe cassette ends; (g) restriction endonuclease sequences flanking thebacterial and yeast sequences to facilitate the removal of thesesequences from the vector.
 5. A method of generating mutations atspecific sites in cloned genomic DNA, comprising the steps of: (a)cloning genomic DNA into the linear lambda vector of claim 1; (b)isolating a clone of interest; (c) converting the linear lambda vectorcontaining the genomic DNA of interest into a circular E. coli/yeastshuttle vector by infecting a Cre recombinase expressing bacterialstrain; (d) identifying genomic DNA sequences intended for targeting ofthe positive selection cassette; (e) synthesizing deoxyoligonucleotidescomplementary to sequences flanking the site intended for targeting ofthe positive selection cassette to said circular E. coli/yeast shuttlevector; (f) attaching said synthetic deoxyoligonucleotides to thepositive selection cassette by ligation or PCR; (g) co-transforming saidE. coli/yeast shuttle vector containing the genomic DNA of interest andthe modified positive selection cassette into a yeast host cell, whereinan intact recombinant plasmid is selected for by means of the geneproducts provided for by yeast selectable markers and is obtained byperforming homologous DNA recombination between homologous regions ofthe vector containing genomic DNA and the synthetically derivedsequences which had been ligated onto the ends of the positive selectioncassette thereby generating a new recombinant vector with the positiveselection cassette inserted into the genomic DNA sequences.
 6. Themethod of generating mutations at specific sites in cloned genomic DNAof claim 5, wherein the E. coli/yeast sequences of the positiveselection cassette vector are removed.
 7. The method of claim 6, whereinsaid removal comprises: (a) digesting said recombinant vector with thepositive selection cassette inserted into the genomic DNA sequences withrestriction endonuclease specific for the unique restrictionendonuclease sequences incorporated into the positive selection cassettevector which flank the E. coli/yeast sequences in said vector; (b)ligating said digested vector; and (c) identifying ligation productslacking the E.coli/yeast sequences desired to be removed.
 8. A method ofproducing mutated animal cells, comprising the steps of: (a) linearizingsaid recombinant vector having a positive selection cassette insertedinto the genomic DNA sequences at a unique restriction endonucleasesequence outside the sequences of the cloned genomic DNA; (b)introducing said linearized DNA into an animal cell; (c) selectingtransduced cells that express said positive selectable marker; and (d)selecting transduced cells that express said positive selectable markerand do not express said negative marker.
 9. The method of claim 8,wherein said animal cell is an embryonic stem cell or any other celltype with the potential to generate an animal.
 10. A method for theproduction of a non-human transgenic animal consisting of: (a)introduction of said mutated animal cells in claim 8 into animalembryos; (b) placement of the embryos containing said mutated cells intothe uterus of a female animal; and (c) replacement of the nucleus of afertilized egg with the nucleus containing the modified genomic DNA.